Not applicable
Not applicable.
This invention is directed to the authentication of Chinese medicinal materials based upon analysis of nucleic acid polymorphisms in an amplified region characteristic of the material in question.
Traditional Chinese medicine has been practiced by the Chinese people for 2-3 millennia. It also currently covers the practice and medicinal materials used by Tibetan, Mongolian and other ethnic minorities. This system with its materials has been spread and adopted by other Asian countries such as Japan, Korea and Vietnam.
Chinese medicinal materials have been recorded in various pharmacopoeia. One of the classical references Bencao Gangmu written by Li Shi Zhen in the late 14th Century contains about 2,500 items of herbs and other products (animals and minerals). The official pharmacopoeia of the People""s Republic of China (1995 ed.) contains 2,375 items of medicinal materials. These medicinal materials are described by their organoleptic characteristics and it is not uncommon to have substitutes or even adulterants in the market. For examples, herbal preparations purporting to contain Panax quinquefolius (American ginseng) may actually contain the less expensive P. ginseng (Asian ginseng). Several poisonous herbs, e.g., Phytolacca acinosa Roxb, Mirabilis jalapa L., and Talnium paniculatum, have been found in medicinals purporting to contain P. ginseng. Similarly, less expensive snakes of different species or even families have been substituted for the three snake crude drugs in the pharmacopoeia, Agkistrodon actus, Bungarus multicinctus multicinctus and Zaocys dhumnades. 
Also, in the market, many medicinal materials exist in powder or shredded slices, rendering their authentication by organoleptic approaches difficult. For the safety of consumers, more effective methods for identification are needed. The presence of these methods also helps the modernization of traditional Chinese medicine and safeguards the healthy development of the medicinal industry. Analytical techniques such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and liquid chromatography/mass spectrometry (LC/MS) may be applied to identify the components in the sample (Lang et al., J. Chi. Pharm. Sci., 2:133-43 (1993)). Nevertheless, the level of the components is easily affected by growing climates, harvesting period and storage conditions. Therefore, for accurate authentication, other independent approaches are needed.
Advances in molecular biology have offered a promising tool and sensitive alternative means for the authentication of medicinal materials. U.S. Pat. No. 5,876,977 discloses the use of polymerase chain reaction-restriction enzyme length polymorphism (PCR-RFLP) analysis to identify herbal materials. It involves first using PCR to amplify a specific region of the genome, and then the appropriate restriction enzyme to cleave the amplified DNA into defined fragments characteristic of the animal or plant medicinal material. However, PCR-RFLP suffers from several disadvantages. Amplification and restriction require two separate enzymatic steps. Moreover, PCR-RFLP depends on the identification of specific restriction site sequence polymorphisms that will yield unique digestion patterns for the particular restriction enzymes chosen.
The present invention employs Random Amplified Polymorphic DNA (RAPD) (Williams et al., Nucleic Acids Res. 18:6531-5 (1990)) and Direct Amplification of Length Polymorphism (DALP) (Desmarais et al., Nucleic Acids Res. 26:1458-65 (1998)) to generate DNA fragments polymorphic to the species under study. Both methods do not require prior genetic knowledge of the sample. In RAPD, a single primer of 10or more nucleotides is used to amplify the genomic DNA. Amplification is under less stringent conditions. In DALP, a pair of primers longer (usually 20-27 bp) than RAPD is used and amplification is performed under more stringent conditions. Both give rise to DNA fragments which produce fingerprints after separation by gel electrophoresis. The origin of these fragments is not defined but they presumably contain DNA sequences complementary to the 3xe2x80x2 end of the primer sequence. Desirable RAPD or DALP polymorphic bands are converted to SCAR (Sequence Characterized Amplified Regions) by first sequencing the band and then designing specific primers for amplification of the specific bands under high stringency conditions.
In one aspect of the invention, an herbal material purporting to contain ginseng is identified by extracting nucleic acids from a sample of the herbal material, amplifying a polymorphic region of the nucleic acid with two or more oligonucleotide primers, resolving the amplification products, and comparing the pattern of amplification products with that from amplification of known herbal materials. In this way, an unknown herbal sample may be identified as Panax ginseng, Panax quinquefolius, Panax notoginseng (Burk), Panax japonicus major, Panax japonicus, Panax trifolius, Mirabilis jalapa L., or Panax acinosa Roxb, or whether the herbal material is from another source.
In another aspect of the invention, a medicinal material purporting to contain extract of a particular snake is identified by extracting nucleic acids from a sample of the medicinal material, amplifying a polymorphic region of the nucleic acid with two or more oligonucleotide primers, resolving the amplification products, and comparing the pattern of amplification products with that from amplification of known snake extracts.
An additional aspect of the invention provides isolated nucleic acids comprising polymorphic regions that may be amplified to identify unknown materials. Still another aspect of the invention provides sets of oligonucleotide primers that amplify polymorphic regions residing between them.
In an embodiment of the invention, the nucleic acid extracted from the herbal or medicinal material is genomic DNA. In an embodiment of the invention, the amplification products are resolved by gel electrophoresis and visualized by fluorescence.
In one embodiment of the invention, the apparent molecular weight of an amplification product from the unknown material is compared with the size of amplification products from known materials to identify the unknown material. In another embodiment, the unknown material is identified by the presence of an amplification product present in the known material, while in yet another embodiment the unknown material is identified by the absence of an amplification product absent from the known material.
In an embodiment of the invention, a polymorphic region of ginseng is first identified by RAPD, employing primer OPC-20. In one embodiment, the polymorphic region identified by DALP comprises a SCAR2 sequence amplified in both P. quinquefolius and P. ginseng, and differing in sequence between various isolates of these species. In a further embodiment, the SCAR2 region is amplified as a SCAR using the primer pair SCAR.F1 and SCAR.R1.
In an embodiment of the invention, a polymorphic region of ginseng is first identified by DALP, employing primers DALP001 and DALPR1. In one embodiment, the polymorphic region identified by DALP analysis comprises the sequence of DALP1.7, amplified only in P. ginseng. In a further embodiment, the DALP1.7 region is amplified as a SCAR using the primer pair DALP1.7F3 and DALP1.7R3.
In an embodiment of the invention, a polymorphic region of ginseng is first identified by RAPD, employing the primer OPC-02. In one embodiment, the polymorphic region identified by RAPD comprises the sequence of C2S8.4, amplified only in P. quinquefolius. In a further embodiment, the C2S8.4 region is amplified as a SCAR using the primer pair C2S8.4F and C2S8.4R.
In an embodiment of the invention, a region polymorphic among different snake species is first identified by RAPD, employing the primer OPF-14. In one embodiment, the region identified by RPD comprises a sequence amplified only in A actus and may be amplified as a SCAR using primer pair SCAR-Af and SCAR-Ar. In another embodiment the region identified by RAPD comprises a sequence amplified only in B. multicinictus multicinctus and may be amplified as a SCAR using primer pair SCAR-Bf and SCAR-Br. In yet another embodiment, the region identified by RAPD comprises a sequence amplified only in Z. dhumnades and may be amplified as a SCAR using primer pair SCAR-Zf and SCAR-Zr.